A novel method for integrating genomic and Tn-Seq data to identify commonin vivofitness mechanisms across multiple bacterial species

Abstract

Sepsis is life-threatening organ dysfunction due to an unregulated immune response to infection. Bacteremia is a leading cause of sepsis, and members of theEnterobacteralescause nearly half of bacteremia cases annually. While previous Tn-Seq studies to identify novel bacteremia-fitness genes have provided valuable insight into virulence mechanisms, evidence for common pathways across species was lacking. To identify common fitness pathways in five bacteremia-causedEnterobacteralesspecies, we utilized the JCVI pan-genome pipeline to integrate Tn-Seq fitness data with multiple available functional data types. Core genes from species pan-genomes were used to construct a multi-species core pan-genome, producing 2,850 core gene clusters found in four out of the five species. Integration of Tn-Seq fitness data enabled identification of 373 protein clusters that were conserved in all five species. A scoring rubric was applied to these clusters, which incorporated Tn-Seq fitness defects, operon localization, and antibiotic susceptibility data to identify seven common bacteremia-fitness pathways. Mutations intatCshowed reduced fitnessin vivoand increased susceptibility to beta-lactams that were restored followingtatCcomplementationin trans. By integrating known operon structures and antibiotic susceptibility with Tn-Seq fitness data, common genes within the core pan-genome emerged and revealed mechanisms that are essential for colonization of, or survival in, the mammalian bloodstream. Our prediction and validation oftatCas a common bacteremia fitness factor and contributor of antibiotic resistance supports the utility of this bioinformatic approach. This study represents a major step forward to identify novel targets of therapy against these deadly widespread sepsis infections.